Michael addition-based probes for ratiometric fluorescence imaging of protein S-depalmitoylases in live cells and tissues

Abstract

The reversible modification of cysteine residues through thioester formation with palmitate (protein S-palmitoylation) is a prevalent chemical modification that regulates the function, localization, and stability of many proteins. Current methods for monitoring the "erasers" of S-palmitoylation, acyl-protein thioesterases (APTs), rely on destructive proteomic methods or "turn-on" probes, precluding deployment in heterogeneous samples such as primary tissues. To address these challenges, we present the design, synthesis, and biological evaluation of Ratiometric Depalmitoylation Probes (RDPs). RDPs respond to APTs with a robust ratiometric change in fluorescent signal both in vitro and in live cells. Moreover, RDPs can monitor endogenous APT activities in heterogeneous primary human tissues such as colon organoids, presaging the utility of these molecules in uncovering novel roles for APTs in metabolic regulation.

Scheme 1. Design of RDPs. A peptide-based S-acylated APT substrate is appended to an aminocoumarin fluorophore. APT activity on the substrate results in a Michael reaction with a cyanoalkene linker, resulting in a blueshift in the probe.

Scheme 2. Synthesis of Ratiometric Depalmitoylation Probe 2 (RDP-2). (i) 20% TFA, DCM, N₂, 1 h. (ii) EDC·HCl, cyanoacetic acid, DMF, 48 h. (iii) Lys(Boc)-OMe·HCl, EDC·HCl, HOBt, DIPEA, DMF, 3 h. (iv) Piperdine, EtOH : DCM 1 : 1, 72 h. (v) I₂, MeOH, 30 min. (vi) TCEP, MeOH. (vii) Octanoic anhydride, Et₃N, DMF. (viii) 15% TFA, DCM.

Fig. 1. UV-Vis absorption (A) and fluorescence emission at λ_ex = 430 nm (B) and λ_ex = 480 nm (C) of 15 μM RDP-2 and deacylated RDP-2 in buffer (20 mM HEPES, 150 mM NaCl, 0.1% Triton X-100, pH 7.4). (D) Ratiometric response of 1 μM RDP-2 to 200 nM APT1 or 200 nM APT2. Error bars are ± std. dev.

Fig. 2. Ratiometric fluorescence imaging of RDP-2 in live HEK293T cells treated with PalmB. (A) Cells were treated with DMSO or 20 μM PalmB for 30 min, loaded with 1 μM RDP-2 for 10 min, and imaged. (B) Quantification of imaging in (A). **p < 0.005. Scale bar = 20 μm. Error bars are ± std. dev. (n = 3).

Fig. 3. Ratiometric fluorescence imaging of RDP-2 in live HEK293T cells treated APT inhibitors. Cells were treated with (A) DMSO or 5 μM ML348 for 30 min and (C) DMSO, 5 μM ML349, or 5 μM ML349 and 5 μM ML348, loaded with 1 μM RDP-2 for 10 min, and imaged. Quantification of (B) ML348 and (D) ML349 with and without ML348 imaging. *p < 0.03, **p < 0.005. Scale bar = 20 μm. Error bars are ± std. dev. (n = 3).

Fig. 4. Ratiometric fluorescence imaging of RDP-2 in live human colon organoids cells treated with PalmB. (A) Organoids were treated with DMSO or 40 μM PalmB for 30 min, loaded with 5 μM RDP-2 for 10 min, and imaged. (B) Quantification of imaging in (A). **p < 0.005. Scale bar = 20 μm. Error bars are ± std. dev. (n = 3).

Fig. 5. Ratiometric fluorescence imaging of RDP-2 in live HEPG2 cells treated with palmitate. (A) Cells were treated with 1% BSA as a control or 1 mM palmitate in 1% BSA for 6 h, loaded with 1 μM RDP-2 for 10 min, and imaged. (B) Quantification of imaging in (A). **p < 0.005. Scale bar = 20 μm. Error bars are ± std. dev. (n = 3).